Method for generating libraries of antibody genes comprising amplification of diverse antibody DNAs and methods for using these libraries for the production of diverse antigen combining molecules

ABSTRACT

A method of producing libraries of genes encoding antigen-combining molecules or antibodies is described. In addition, a method of producing antigen-combining molecules which does not require an in vivo procedure is described. Vectors useful in the present method and antigen-combining molecules produced by the method are discussed. The antigen-combining molecules are useful for the detection, quantitation, purification and neutralization of antigens, as well as for diagnostic, therapeutic and prophylactic purposes.

This application is a continuation of prior application Ser. No.09/439,732, filed Nov. 12, 1999 now U.S. Pat. No. 6,303,313, which is acontinuation of prior application Ser. No. 08/997,195, filed on Dec. 23,1997 now abandon, which is a continuation of prior application Ser. No.08/315,269, filed on Sep. 29, 1994 now U.S. Pat. No. 5,780,225, which isa continuation of prior application Ser. No. 07/919,730, filed on Jul.24, 1992 now U.S. Pat. No. 5,284,555, which is a continuation of priorapplication Ser. No. 07/464,350, filed on Jan. 11, 1990 now abandoned.

BACKGROUND OF THE INVENTION

Monoclonal and polyclonal antibodies are useful for a variety ofpurposes. The precise antigen specificity of antibodies makes thempowerful tools that can be used for the detection, quantitation,purification and neutralization of antigens.

Polyclonal antibodies are produced in vivo by immunizing animals, suchas rabbits and goats, with antigens, bleeding the animals and isolatingpolyclonal antibody molecules from the blood. Monoclonal antibodies areproduced by hybridoma cells, which are made by fusing, in vitro,immortal plasmacytoma cells with antibody producing cells (Kohler, G.and C. Milstein, Nature, 256:495 (1975)) obtained from animals immunizedin vivo with antigen.

Current methods for producing polyclonal and mono-clonal antibodies arelimited by several factors. First, methods for producing eitherpolyclonal or monoclonal antibodies require an in vivo immunizationstep. This can be time consuming and require large amounts of antigen.Second, the repertoire of antibodies expressed in vivo is restricted byphysiological processes, such as those which mediate self-tolerance thatdisable-auto-reactive B cells (Goodnow, C. C., et al., Nature, 334:676(1988); Goodnow, J. W., Basic and Clinical Immunology, Ed. 5, Los Altos,Calif., Large Medical Publications (1984); Young, C. R., MolecularImmunology, New York, Marcel Dekker (1984)). Third, although antibodiescan exist in millions of different forms, each with its own uniquebinding site for antigen, antibody diversity is restricted by geneticmechanisms for generating antibody diversity (Honjo, T., Ann. Rev.Immunol., 1:499 (1983); Tonegawa, S., Nature:302:575 (1983)). Fourth,not all the antibody molecules which can be generated will be generatedin a given animal. As a result, raising high affinity antibodies to agiven antigen can be very time consuming and can often fail. Fifth, theproduction of human antibodies of desired specificity is veryproblematical.

A method of producing antibodies which avoids the limitations ofpresently-available methods, such as the requirement for immunization ofan animal and in vivo steps, would be very useful, particularly if itmade it possible to produce a wider range of antibody types than can bemade using presently-available techniques and if it made it possible toproduce human antibody types.

DISCLOSURE OF THE INVENTION

The present invention relates to a method of producing libraries ofgenes encoding antigen-combining molecules or antibodies; a method ofproducing antigen-combining molecules, also referred to as antibodies,which does not require an in vivo procedure, as is required bypresently-available methods; a method of obtaining antigen-combiningmolecules (antibodies) of selected or defined specificity which does notrequire an in vivo procedure; vectors useful in the present method andantibodies produced or obtained by the method.

The present invention relates to an in vitro process for synthesizingDNA encoding families of antigen-combining molecules or proteins. Inthis process, DNA containing genes encoding antigen-combining moleculesis obtained and combined with oligonucleotides which are homologous toregions of the genes which are conserved. Sequence-specific geneamplification is then carried out using the DNA containing genesencoding antigen-combining proteins as template and the homologousoligonucleotides as primers.

This invention also relates to a method of creating diverse libraries ofDNAs encoding families of antigen-combining proteins by cloning theproduct of the in vitro process for synthesizing DNA, described in thepreceeding paragraph, into an appropriate vector (e.g., a plasmid, viralor retroviral vector).

The subject invention provides an alternative method for the productionof antigen-combining molecules, which are useful affinity reagents forthe detection and neutralization of antigens and the delivery ofmolecules to antigenic sites. The claimed method differs from productionof polyclonal antibody molecules derived by immunization of live animalsand from production of mono-clonal antibody molecules through the use ofhybridoma cell lines in that it does not require an in vivo immunizationstep, as do presently available methods. Rather, diverse libraries ofgenes which encode antigen-combining sites comprising a significantproportion of an animal's repertoire of antibody combining sites aremade, as described in detail herein. These genes are expressed in livingcells, from which molecules of desired antigenic selectivity can beisolated and purified for various uses.

Antigen-combining molecules are produced by the present method in thefollowing manner, which is described in greater detail below. Initially,a library of antibody genes which includes a set of variable regionsencoding a large, diverse and random group of specificities derived fromanimal or human immunoglobulins is produced by amplifying or cloningdiverse genomic fragments or cDNAs of antibody mRNAs found inantibody-producing tissue.

In an optional step, the diversity of the resulting libraries can beincreased by means of random mutagenesis. The gene libraries areintroduced into cultured host cells, which may be eukaryotic orprokaryotic, in which they are expressed. Genes encoding antibodies ofdesired antigenic specificity are identified, using a method describedherein or known techniques, isolated and expressed in quantities inappropriate host cells, from which the encoded antibody can be purified.

Specifically, a library of genes encoding immunoglobulin heavy chainregions and a library of genes encoding immunoglobulin light chainregions are constructed. This is carried out by obtainingantibody-encoding DNA, which is either genomic fragments or cDNAs ofantibody mRNAs, amplfying or cloning the fragments or cDNAs; andintroducing them into a standard framework antibody gene vector, whichis used to introduce the antibody-encoding DNA into cells in which theDNA is expressed. The vector includes a framework gene encoding aprotein, such as a gene encoding an antibody heavy chain or an antibodylight chain which can be of any origin (human, non-human) and can bederived from any of a number of existing DNAs encoding heavy chainimmunoglobulins or light chain immunoglobulins. Such vectors are also asubject of the present invention and are described in greater detail ina subsequent section. Genes from one or both of the libraries areintroduced into appropriate host cells, in which the genes areexpressed, resulting in production of a wide variety ofantigen-combining molecules.

Genes encoding antigen-combining molecules of desired specificity areidentified by identifying cells producing antigen-combining moleculeswhich react with a selected antigen and then obtaining the genes ofinterest. The genes of interest can subsequently be introduced into anappropriate host cell (or can be further modified and then introducedinto an appropriate host cell) for further production ofantigen-combining molecules, which can be purified and used for the samepurposes for which conventionally-produced antibodies are used.

Through use of the method described, it is possible to produceantigen-combining molecules which are of wider diversity than areantibodies available as a result of known methods; novelantigen-combining molecules with a diverse range of specificities andaffinities and antigen-combining molecules which are predominantly humanin origin. Such antigen-combining molecules are a subject of the presentinvention and can be used clinically for diagnostic, therapeutic andprophylactic purposes, as well as in research contexts, and for otherpurposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the method of the presentinvention by which antigen-combining molecules, or antibodies, areproduced.

FIG. 2 is a schematic representation of amplification or cloning of IgMheavy chain variable region DNA from mRNA, using the polymerase chainreaction.

Panel A shows the relevant regions of the poly adenylated mRNA encodingthe secreted form of the IgM heavy chain denotes the sequences encodingthe signal peptide which causes the nascent peptide to cross the plasmamembrane. V, D and J together comprise the variable region. C_(H)1,C_(H)2, and C_(H)3 are the three constant domains of Cμ. Hinge encodesthe hinge region. C, B and Z are oligonucleotide PCR primers (discussedbelow).

Panel B shows the reverse transcript DNA product of the mRNA primed byoligonucleotide Z, with the addition of poly-dC by terminal transferaseat the 3′ end.

Panel C is a schematic representation of the annealing of primer A tothe reverse transcript DNA.

Panel D shows the final double stranded DNA PCR product made utilizingprimers A and B.

Panel E shows the product of PCR annealed to primer C.

Panel F is a blowup of Panel E, showing in greater detail the structureof primer C. Primer C consists of two parts: a 3′ part complementary toIgM heavy chain mRNA as shown, and a 5′ part which contains restrictionsite RE2 and spacer.

Panel G shows the final double stranded DNA PCR product made utilizingprimers A and C and the product of the previous PCR (depicted in D) astemplate. The S, V, D, J regions are again depicted.

FIG. 3 is a schematic representation of the heavy chain framework vectorpFHC. The circular plasmid (above) is depicted linearized (below) andits relevant components are shown: animal cell antibiotic resistancemarker; bacterial replication origin; bacterial cell antibioticresistance marker; Cμ enhancer; LTR containing the viral promoter fromthe Moloney MLV retrovirus DNA; PCR primer (D); cDNA cloning sitecontaining restriction endonuclease sites, RE1 and RE2, separated byspacer DNA; Cμ exons; and poly A addition and termination sequencesderived from the Cμ gene or having the same sequence as the Cμ gene.

FIG. 4 depicts a nucleotide sequence of the C_(H)1 exon of the Cμ gene,and its encoded amino acid sequence (Panel A). The nucleotide coordinatenumbers are listed above the line of nucleotide sequences. Panel Bdepicts the N-doped sequence, as defined in the text.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of producing antigen-combiningmolecules (or antibodies) which does not require an in vivo immunizationprocedure and which makes it possible to produce antigen-combiningmolecules with far greater diversity than is shown by antibodiesproduced by currently-available techniques.

The present invention relates to a method of producing libraries ofgenes encoding antigen-combining molecules (antibody proteins) withdiverse antigen-combining specificities; a method of producing suchantigen-combining molecules, antigen-combining molecules produced by themethod and vectors useful in the method. The following is a descriptionof generation of such libraries, of the present method of producingantigen-combining molecules of selected specificity and of vectorsuseful in producing antigen-combining molecules of the presentinvention.

As described below, the process makes use of techniques which are knownto those of skill in the art and can be applied as described herein toproduce and identify antigen-combining molecules of desired antigenicspecificity: the polymerase chain reaction (PCR), to amplify and clonediverse cDNAs encoding antibody mRNAs found in antibody-producingtissue; mutagenesis protocols to further increase the diversity of thesecDNAs; gene transfer protocols to introduce antibody genes into cultured(prokaryotic and eukaryotic) cells for the purpose of expressing them;and screening protocols to detect genes encoding antibodies of thedesired antigenic specificity. A general outline of the present methodis represented in FIG. 1.

Construction of Library of Genes Encoding Antigen-Combining Molecules

A key step in the production of antigen-combining molecules by thepresent method is the construction of a “library” of antibody geneswhich include “variable” regions encoding a large, diverse, but randomset of specificities. The library can be of human or non-human originand is constructed as follows:

Initially, genomic DNA encoding antibodies or cDNAs of antibody mRNA(referred to as antibody-encoding DNA) is obtained. This DNA can beobtained from any source of antibody-producing cells, such as spleencells, peripheral blood cells, lymph nodes, inflammatory tissue cellsand bone marrow cells. It can also be obtained from a genomic library orcDNA library of B cells. The antibody-producing cells can be of human ornon-human origin; genomic DNA or mRNA can be obtained directly from thetissue (i.e., without previous treatment to remove cells which do notproduce antibody) or can be obtained after the tissue has been treatedto increase concentration of antibody-producing cells or to select aparticular type(s) of antibody-producing cells (i.e., treated to enrichthe content of antibody-producing cells). Antibody-producing cells canbe stimulated by an agent which stimulates antibody mRNA production(e.g., lipopolysaccharide) before DNA is obtained.

Antibody-encoding DNA is amplified and cloned using a known technique,such as the PCR using appropriately-selected primers, in order toproduce sufficient quantities of the DNA and to modify the DNA in such amanner (e.g., by addition of appropriate restriction sites) that it canbe introduced as an insert into an E. coli cloning vector. This cloningvector can serve as the expression vector or the inserts can later beintroduced into an expression vector, such as the framework antibodygene vector described below. Amplified and cloned DNA can be furtherdiversified, using mutagenesis, such as PCR, in order to produce agreater diversity or wider repertoire of antigen-binding molecules, aswell as novel antigen-binding molecules.

Cloned antibody-encoding DNA is introduced into an expression vector,such as the framework antibody gene vector of the present invention,which can be a plasmid, viral or retroviral vector. Clonedantibody-encoding DNA is inserted into the vector in such a manner thatthe cloned DNA will be expressed as protein in appropriate host cells.It is essential that the expression vector used make it possible for theDNA insert to be expressed as a protein in the host cell. One expressionvector useful in the present method is referred to as the frameworkantibody gene vector. Vectors useful in the present method containantibody constant region or portions thereof in such a manner that whenamplified DNA is inserted, the vector expresses a chimeric gene productcomprising a variable region and a constant region in proper register.The two regions present in the chimeric gene product can be from thesame type of immunoglobulin molecule or from two different types ofimmunoglobulin molecules.

These libraries of antibody-encoding genes are then expressed incultured cells, which can be eukaryotic or prokaryotic. The librariescan be introduced into host cells separately or together. Introductionof the antibody-encoding DNA in vitro into host cells (by infection,transformation or transfection) is carried out using known techniques,such as electroporation, protoplast fusion or calcium phosphateco-precipitation. If only one library is introduced into a host cell,the host cell will generally be one which makes the other antibodychain, thus making it possible to produce complete/functionalantigen-binding molecules. For example, if a heavy chain libraryproduced by the present method is introduced into host cells, the hostcells will generally be cultured cells, such as myeloma cells or E.coli, which naturally produce the other (i.e., light) chain of theimmunoglobulin or are engineered to do so. Alternatively, both librariescan be introduced into appropriate host cells, either simultaneously orsequentially.

Host cells in which the antibody-encoding DNA is expressed can beeukaryotic or prokaryotic. They can be immortalized cultured animalcells, such as a myeloma cell line which has been shown to efficientlyexpress and secrete introduced immunoglobulin genes (Morrison, S. L., etal., Ann. N.Y. Acad. Sci., 507:187 (1987); Kohler, G. and C. Milstein,Eur. J. Immunol., 6:511 (1976); Oi, V. T., et al., Immunoglobulin GeneExpression in Transformed Lymphoid Cells, 80:825 (1983); Davis, A. C.and M. J. Shulman, Immunol. Today, 10:119 (1989)). One host cell whichcan be used to express the antibody-encoding DNA is the J558L cell lineor the SP2/0 cell line.

Cells expressing antigen-combining molecules with a desired specificityfor a given antigen can then be selected by a variety of means, such astesting for reactivity with a selected antigen using nitrocelluloselayering. The antibodies identified thereby can be of human origin,nonhuman origin or a combination of both. That is, all or some of thecomponents (e.g., heavy chain, light chain, variable regions, constantregions) can be encoded by DNA of human or nonhuman origin, which, whenexpressed produces the encoded chimeric protein which, in turn, may behuman, nonhuman or a combination of both. In such antigen-combiningmolecules, all or some of the regions (e.g., heavy and light chainvariable and constant regions) are referred to as being of human originor of nonhuman origin, based on the source of the DNA encoding theantigen-combining molecule region in question. For example, in the casein which DNA encoding mouse heavy chain variable region is expressed inhost cells, the resulting antigen-combining molecule has a heavy chainvariable region of mouse origin. Antibodies produced may be used forsuch purposes as drug delivery, tumor imaging and other therapeutic,diagnostic and prophylactic uses.

Once antibodies of a desired binding specificity are obtained, theirgenes may be isolated and further mutagenized to create additionalantigen combining diversity or antibodies of higher affinity forantigen.

Construction of Immunoglobulin Heavy Chain Gene Library and Productionof Encoded Antigen-Binding Molecules

The following is a detailed description of a specific experimentalprotocol which embodies the concepts described above. Although thefollowing is a description of one particular embodiment, the sameprocedures can be used to produce libraries in which the immunoglobulinand the heavy chain class are different or in which light chain genesare amplified and cloned. The present invention is not intended to belimited to this example. In the embodiment presented below, a diverseheavy chain gene library is constructed. Using the principles describedin relation to the heavy chain gene library, a diverse light chain genelibrary is also constructed. These are co-expressed in an immortal tumorcell capable of producing antibodies, such as plasmacytoma cells ormyeloma cells. Cells expressing antibody reactive to antigen areidentified by a nitrocellulose filter overlay and antibody is preparedfrom cells identified as expressing it. As described in a subsequentsection, there are alternative methods of library construction, otherexpression systems which can be used, and alternative selection systemsfor identifying antibody-producing cells or viruses.

Step 1 in this specific protocol is construction of libraries of genesin E. coli which encode immunoglobulin heavy chains. This is followed bythe use of random mutagenesis to increase the diversity of the library,which is an optional procedure. Step 2 is introduction of the library,by transfection, into myeloma cells. Step 3 is identification of myelomacells expressing antibody with the desired specificity, using thenitrocellulose filter overlay technique or techniques known to those ofskill in the art. Step 4 is isolation of the gene(s) encoding theantibody with the desired specificity and their expression inappropriate host cells, to produce antigen-combining fragments usefulfor a variety of purposes.

Construction

One key step in construction of the library of cDNAs encoding thevariable region of mouse heavy chain genes is construction of an E. coliplasmid vector, designated pFHC. pFHC contains a “framework” gene, whichcan be any antibody heavy chain and serves as a site into which theamplified cloned gene product (genomic DNA or cDNA of antibody mRNAs) isintroduced. pFHC is useful as a vector for this purpose because itcontains RE1 and RE2 cloning sites. Other vectors which include aframework gene and other cloning sites can be used for this purpose aswell. The framework gene includes a transcriptional promoter (e.g., apowerful promoter, such as a Moloney LTR (Mulligan, R. C., InExperimental Manipulation of Gene Expression, New York Adacemic Press,p. 155 (1983)) and a Cμ chain transcriptional enhancer to increase thelevel of transcriptions from the promoter (Gillies, S. D., et al., Cell,33:717 (1983), a cloning site containing RE1 and RE2; part of the Cμheavy chain gene encoding secreted protein; and poly A addition andtermination sequences (FIG. 3). The framework antibody gene vector ofthe present invention (pFHC) also includes a selectable marker (e.g., anantibiotic resistance gene such as the neomycin resistance gene,neo^(R)) for animal cells; sequences for bacterial replication (ori);and a selectable marker (e.g., the ampicillin resistance gene, Amp^(R))for bacterial cells. The framework gene can be of any origin (human,non-human), and can derive from any one of a number of existing DNAsencoding heavy chain immunoglobulins (Tucker, P. W., et al., Science,206:1299 (1979); Honjo, T., et al., Cell, 18:559 (1979); Bothwell, A. L.M., et al., Cell, 24:625 (1981); Liu, A. Y, et al., Gene, 54:33 (1987);Kawakami, T., et al., Nuc. Acids. Res., 8:3933 (1980)). In thisembodiment, the vector retains the introns between the C_(H)1, hinge,C_(H)2 and C_(H)3 exons. The “variable region” of the gene, whichincludes the V, D and J regions of the antibody heavy chain and whichencodes the antigen binding site, is deleted and replaced with twoconsecutive restriction endonuclease cloning sites, RE1 and RE2. Therestriction endonuclease site RE1 occurs just 3′ to the LTR promoter andthe restriction endonuclease site RE2 occurs within the constant regionjust 3′ to the J region (see FIG. 3).

Another key step in the production of antigen-combining molecules inthis embodiment of the present invention is construction in an E. colivector of a library of cDNAs encoding the variable region of mouseimmunoglobulin genes. In this embodiment, the pFHC vector, whichincludes cloning sites designated RE1 and RE2, is used for cloning heavychain variable regions, although any cloning vector with cloning siteshaving the same or similar characteristics (described below) can beused. Similarly, a light chain vector can be designed, using the abovedescribed procedures and procedures known to a person of ordinary skillin the art.

In this embodiment, non-immune mouse spleens are used as the startingmaterial. mRNA is prepared directly from the spleen or from spleenprocessed in such a manner that it is enriched for resting B cells.Enrichment of tissue results in a more uniform representation ofantibody diversity in the starting materials. Lymphocytes can bepurified from spleen using ficoll gradients (Boyum, A., Scand. J. ofClinical Invest., 21:77 (1968)). B cells are separated from other cells(e.g., T cells) by panning with anti-IgM coated dishes (Wysocki, L. J.and V. L. Sato, Proc. Natl. Acad. Sci., 75:2844 (1978)). Becauseactivated cells express the IL-2 receptor but resting B cells do not,resting B cells can be separated yet further from activated cells bypanning. Further purification by size fractionation on a Cell Sorterresults in a fairly homogeneous population of resting B cells.

Poly A+ mRNA from total mouse spleen is prepared according to publishedmethods (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual,2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)). Production of antibody mRNA can first be stimulated bylipopolysaccharide (LPS) (Andersson, J. A., et al., J. Exp. Med.,145:1511 (1977)). First strand cDNA is prepared to this mRNApopulation-using as primer an oligonucleotide, Z, which is complementaryto Cμ in the C_(H)1 region 3′ to J. This primer is designated Z in FIG.2. First strand cDNA is then elongated by the terminal transferasereaction with dCTP to form a poly dC tail (Sambrook, J., et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)).

This DNA product is then used as template in a polymerase chain reaction(PCR) to amplify cDNAs encoding antibody variable regions (Saiki, R. K.,et al., Science, 239:487 (1988); Ohara, O., et al., Proc. Natl. Acad.Sci. USA, 86:5673 (1989)). Initially, PCR is carried out with twoprimers: primer A and primer B, as represented in FIG. 2. Primer Acontains the RE1 site at its 5′ end, followed by poly dG. Primer B iscomplementary to the constant (C_(H)1) region of the Cμ gene, 3′ to theJ region and 5′ to primer Z (see FIG. 2). Primer B is complementary toall Cμ genes, which encode the heavy chain of molecules of the IgMclass, the Ig class expressed by all B cell clones prior to classswitching (Schimizu, A. and T. Honjo, Cell, 36:801-803 (1984)) andpresent in resting B cells. The resultant PCR product includes asignificant proportion of cDNAs encompassing the various V_(H) regionsexpressed as IgM in the mouse. (The use of other primers complementaryto the cDNA genes encoding the constant regions of other immunoglobulinheavy chains can be used in parallel reactions to obtain the variableregions expressed on these molecules, but for simplicity these are notdescribed).

Next, the product of the first PCR procedure is used again for PCR withprimer A and primer C. Primer C, like primer B, is complementary to theCμ gene 3′ to J and just 5′ to primer B (see FIG. 2). Primer C containsthe RE2 site at its 5′ end. The RE2 sequence is chosen in such a mannerthat when it is incorporated into the framework vector, no alteration ofcoding sequence of the Cμ chain occurs (See FIGS. 2 and 3). This methodof amplifying Cμ cDNAs, referred to as unidirectional nested PCR,incorporates the idea of nested primers for cloning a gene when thenucleotide sequence of only one region of the gene is known (Ohara, O.,et al., Proc. Natl. Acad. Sci. USA, 86:5673 (1989)). The PCR product isthen cleaved with restriction enzymes RE1 and RE2 and cloned into theRE1 and RE2 sites of the pFHC vector (described below). The sequence ofprimers and of RE1 and RE2 sites are selected so that when the PCRproduct is cloned into these sites, the sites are recreated and thecloned antibody gene fragments are brought back into the proper framewith respect to the framework immunoglobulin gene present in pFHC. Thisresults in creation of a Cμ minigene which lacks the intron normallypresent between J and the C_(H)1 region of Cμ (See FIG. 3). Theseprocedures result in production of the heavy chain library used toproduce antigen-binding molecules of the present invention, as describedfurther below.

Optionally, diversity of the heavy chain variable region is increased byrandom mutagenesis, using techniques known to those of skill in the art.

For example, the library produced as described above is amplified again,using PCR under conditions of limiting nucleotide concentration. Suchconditions are known to increase the infidelity of the polymerizationand result in production of mutant products. Primers useful for thisreaction are Primers C and D, as represented in FIGS. 2 and 3. Primer Dderives from pFHC just 5′ to RE1. The PCR product, after cleavage withRE1 and RE2, is recloned into the framework vector pFHC. To the extentthat mutation affects codons of the antigen binding region, thisprocedure increases the diversity of the binding domains. For example,if the starter library has a complexity of 10⁶ elements, and an averageof one mutation is introduced per complementarity determining region,and it is assumed that the complementarity determining region is 40amino acids in size and that any of six amino acid substitutions canoccur at a mutated codon, the diversity of the library can be increasedby a factor of about 40×6, or 240, for single amino acid changes and240×240, or about 6×10⁴, for double amino acid changes, yielding a finaldiversity of approximately 10¹¹. This is considered to be in the rangeof the diversity of antibodies which animals produce (Tonegawa, S.,Nature, 302:575 (1983)). Even greater diversity can be generated by therandom combination of H and L chains, the result of co-expression inhost cells (see below). It is, thus, theoretically possible to generatea more diverse antibody library in vitro than can be generated in vivo.This library of genes is called the “high diversity” heavy chainlibrary. It may be propagated indefinitely in E. coli. A high diversitylight chain library can be prepared similarly.

The framework vector for the light chain library, designated pFLC,includes components similar to those in the vector for the heavy chainlibrary: the enhancer, promoter, a bacterial selectable marker, ananimal selectable marker, bacterial origin of replication and lightchain exons encoding the constant regions. For pFLC, the animalselectable marker should differ from the animal selectable in pFHC. Forexample, if pFHC contains neo^(R), pFLC can contain Eco gpt.

A light chain library, which contains diverse light chain fragments, isprepared as described above for construction of the heavy chain library.In constructing the light chain library, the primers used are differentfrom those described above for heavy chain library construction. In thisinstance, the primers are complementary to light chain mRNA encodingconstant regions. The framework vector contains the light chain constantregion exons.

Introduction of the Library of Immunoglobulin Chain Genes intoImmortalized Animal Cells

The library of immunoglobulin chain genes produced as described issubsequently introduced into a line of immortalized cultured animalcells, referred to as the “host” cells, in which the genes in thelibrary are expressed. Particularly useful for this purpose areplasmacytoma cell lines or myeloma cell lines which have been shown toefficiently express and secrete introduced immunoglobulin genes(Morrison, S. L., et al., Ann. N.Y. Acad. Sci., 507:187 (1987); Kohler,G. and C. Milstein, Eur. J. Immunol., 6:511 (1976); Galfre and C.Milstein, Methods Enzymol., 73:3 (1981); Davis, A. C. and M. J. Shulman,Immunol. Today, 10:119 (1989)). For example, the J558L cell line can becotransfected using electroporation or protoplast fusion (Morrison, S.L., et al., Ann. N.Y. Acad. Sci., 507:187 (1987)) and transfected cellsselected on the basis of auxotrophic markers present on light and heavychain libraries.

As a result of cotransformation and selection for markers on both lightchain and heavy chain vectors, most transformed host cells will expressseveral copies of immunoglobulin heavy and light chains from the diverselibrary, and will express chimeric antibodies (antibodies encoded by allor part of two or more genes) (Nisonoff, A., et al., In The AntibodyMolecule, Academic Press, NY p. 238 (1975)). These chimeric antibodiesare of two types: those in which one chain is encoded by a host cellgene and the other chain is encoded by an exogenously introducedantibody gene and those in which both the light and the heavy chain areencoded by an exogenous antibody gene. Both types of antibodies will besecreted. A library of cells producing antibodies of diversespecificities is produced as a result. The library of cells can bestored and maintained indefinitely by continuous culture and/or byfreezing. A virtually unlimited number of cells can be obtained by thisprocess.

Isolation of Cells Producing Antigen-Binding Molecules of SelectedSpecificity

Cells producing antigen-binding molecules of selected specificity (i.e.,which bind to a selected antigen) can be identified and isolated usingnitrocellulose filter layering or known techniques. The same methodsemployed to identify and isolate hybridoma cells producing a desiredantibody can be used: cells are pooled and the supernatants tested forreactivity with antigen (Harlow, E. and D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y., p. 283 (1988).Subsequently, individual clones of cells are identified, using knowntechniques. A preferred method for identification and isolation of cellsmakes use of nitrocellulose filter overlays, which allow the screeningof a large number of cells. Cells from the library of transfectedmyeloma cells are seeded in 10 cm² petri dishes in soft agar (Cook, W.D. and M. D. Scharff, PNAS, 74:5687 (1977); Paige, C. J., et al.,Methods in Enzymol., 150:257 (1987)) at a density of 10⁴ colony formingunits, and allowed to form small colonies (approximately 300 cells). Alarge number of dishes (>100) may be so seeded. Cells are then overlayedwith a thin film of agarose (<1 mm) and the agarose is allowed toharden. The agarose contains culture medium without serum.Nitrocellulose filters (or other protein-binding filters) are layered ontop of the agarose, and the dishes are incubated overnight. During thistime, antibodies secreted by the cells will diffuse through the agaroseand adhere to the nitrocellulose filters. The nitrocellulose filters arekeyed to the underlying plate and removed for processing.

The method for processing nitrocellulose filters is identical to themethods used for Western blotting (Harlow, E. and D. Lane, Antibodies:Laboratory Manual, Cold Spring Harbor, N.Y., p. 283 (1988)). Theantibody molecules are adsorbed to the nitrocellulose filter. Thefilters, as prepared above, are then blocked. The desired antigen, forexample, keyhole lymphet hemocyanin (KLH), which has been iodinated withradioactive ¹²⁵I, is then applied in Western blotting buffers to thefilters. (Other, non radiographic methods can be used for detection).After incubation, the filters are washed and dried and used to exposeautoradiography film according to standard procedures. Where the filtershave adsorbed antibody molecules which are capable of binding KLH, theautoradiography film will be exposed. Cells expressing the KLH reactiveantibody can be identified by determining the location on the dishcorresponding to an exposed filter; cells identified in this manner canbe isolated using known techniques. Cells which are isolated from aregion of the dish can then be rescreened, to insure the isolation ofthe clone of antigen-binding molecule-producing cells.

Isolation of Genes Encoding Antigen-Binding Molecules of SelectedSpecificity and Purification of Encoded Antigen-Binding Molecules

The gene(s) encoding an antigen-binding molecule of selected specificitycan be isolated. This can be carried out, for example, as follows:primers D and C (see FIGS. 2 and 3) are used in a polymerase chainreaction, to produce all the heavy chain variable region genesintroduced into the candidate host cell from the library. These genesare cloned again in the framework vector pFHC at the RE1 and RE2 sitesSimilarly, all the light chain regions introduced into the host cellfrom the library are cloned into the light chain vector, pFLC. Membersof the family of vectors so obtained are then transformed pairwise intomyeloma cells, which are tested for the ability to produce and secretethe antibody with the desired selectivity. Purification of the antibodyfrom these cells can then be accomplished using standard procedures(Johnstone, A. and R. Thorpe, Immunochem. in Practice, BlackwellScientific, Oxford, p. 27 (1982); Harlow, E. and D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y., p. 283 (1988)).

Alteration of Affinity of Antigen-Binding Molecules

It is also possible to produce antigen-binding molecules whose affinityfor a selected antigen is altered (e.g., different from the affinity ofa corresponding antigen-binding molecule produced by the presentmethod). This can be carried out, for example, to increase the affinityof an antigen-binding molecule by randomly mutagenizing the genesisolated as described above using previously-described mutagenesismethods. Alternatively, the variable region of antigen-bindingmolecule-encoding genes can be sequenced and site directed mutagenesisperformed to mutate the complementarity determining regions (CDR)(Kabat, E. A., J. Immunol., 141:S 25-36 (1988)). Both processes resultin production of a sublibrary of genes which can be screened forantigen-binding molecules of higher affinity or of altered affinityafter the genes are expressed in myeloma cells.

Alternative Materials and Procedures for Use in the Present Method

In addition to those described above for use in the method of thepresent invention, other materials (e.g., starting materials, primers)and procedures can be used in carrying out the method. For example, useof PCR technology to clone a large collection of cDNA genes encodingvariable regions of heavy chains has been described above. Althoughprimers from the Cμ class were described as being used in unidirectionalnested PCR, the present invention is not limited to these conditions.For example, primers from any of the other heavy chain classes (Cγ₃,Cγ₁, Cγ_(2b), Cα for example) or from light chains can be used. Cμ wasdescribed as of particular use because of the fact that the entirerepertoire of heavy chain variable regions are initially expressed asIgM. Only following heavy-chain class switching are these variableregions expressed with a heavy chain of a different class (Shimizu, A.and T. Honjo, Cell, 36:801-803 (1984)). In addition, the predominantpopulation of B cells in nonimmune spleen cells is IgM⁺-cells (Cooper,M. D. and P. Burrows, In Immunoglobulin Genes, Academic Press, N.Y. p. 1(1989)). Although unidirectional nested PCR amplification is describedabove, other PCR procedures, as well as other DNA amplificationtechniques can be used to amplify DNA as needed in the presentinvention. For example, bidirectional PCR amplification of antibodyvariable regions can be carried out. This approach requires use ofmultiple degenerate 5′ primers (Orlandi, R., et al., Proc. Natl. Acad.Sci. USA, 86:3833 (1989); Sastry, L., et al., Proc. Natl. Acad. Sci.USA, 86:5728 (1989)). Bidirectional amplification may not pick up thesame full diversity of genes as can be expected from unidirectional PCR.

In addition, methods of introducing further diversity into the antibodylibrary other than the method for random mutagenesis utilizing PCRdescribed above can be used. Other methods of random mutagenesis, suchas that described by Sambrook, et al. (Sambrook, J., et al., MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)) can be used, as can direct mutagenesis ofthe complementarity determining regions (CDRs).

Framework vectors other than one using a mouse Cμ heavy chain constantregion, which contains the Cμ enhancer and introns and a viral promoter(described previously) can be used for inserting the products of PCR.The vectors described were chosen for their subsequent use in theexpression of the antibody genes, but any eukaryotic or prokaryoticcloning vector could be used to create a library of diverse cDNA genesencoding variable regions of antibody molecules. The inserts from thisvector could be transferred to any number of expression vectors. Forexample, other framework vectors which include intronless genes can beconstructed, as can other heavy chain constant regions. In addition toplasmid vectors, viral vectors or retroviral vectors can be used tointroduce genes into myeloma cells.

The source for antibody molecule mRNAs can also be varied. Purifiedresting B lymphocytes from mouse nonimmunized spleen are described aboveas such a source. However, total spleens (immunized or not) from otheranimals, including humans, can be used, as can any source ofantibody-producing cells (e.g., peripheral blood, lymph nodes,inflammatory tissue, bone marrow).

Introduction of H and L chain gene DNA into myeloma cells usingcotransformation by electroporation or protoplast fusion methods isdescribed above (Morrison, S. L. and V. T. Oi, Adv. Immunol., 44:65(1989)). However, any means by which DNA can be introduced into livingcells in vivo can be used, provided that it does not significantlyinterfere with the ability of the transformed cells to express theintroduced DNA. In fact, a method other than cotransformation, can beused. Cotransfection was chosen for its simplicity, and because both theH and L chains can be introduced into myeloma cells. It may be possibleto introduce only the H chain into myeloma cells. Moreover, the H chainitself in many cases carries sufficient binding affinity for antigen.However, other methods can also be used. For example, retroviralinfection may be used. Replication-incompetent retroviral vectors can bereadily constructed which can be packaged into infective particles byhelper cells (Mann, R., et al., Cell, 33:153-159 (1903)). Viral titersof 10⁵ infectious units per ml. can be achieved, making possible thetransfer of very large numbers of genes, into myeloma cells.

Further increases in the diversity of antibody-producing cells thanresults from the method described above can be generated if light andheavy chain genes are introduced separately into myeloma cells. Lightchain genes can be introduced into one set of myeloma cells with oneselectable marker, and heavy chains into another set of cells with adifferent selectable marker. Myeloma cells containing and expressingboth H and L chains could then be generated by the highly efficientprocess of polyethylene glycol mediated cell fusion (Pontecorvo, G.,Somatic Cell Genetics, 1:397 (1975)). Thus, a method of screeningdiverse libraries of antibody genes using animal cells is not limited bythe number of cells which can be generated, but by the number of cellswhich can be screened.

Methods of identifying antigen-binding molecule-expressing cellsexpressing an antigen-binding molecule of selected specificity otherthan the nitrocellulose filter overlay technique described above can beused. An important characteristic of any method is that it be useful toscreen large numbers of different antibodies. With the nitrocellulosefilter overlay technique, for example, if 300 dishes are prepared and10⁴ independent transformed host cells per dish are screened, and if, onaverage, each cell produces ten different antibody molecules, then300×10⁴×3, or about 10⁷ different antibodies can be screened at once.However, if the antibody molecules can be displayed on the cell surface,still larger numbers of cells can be screened using affinity matrices topre-enrich for antigen-binding cells. There are immortal B cell lines,such as BCL₁B₁, which will express IgM both on the cell surface and as asecreted form (Granowicz, E. S., et al., J. Immunol., 125:976 (1980)).If such cells are infected by retroviral vectors containing the terminalCμ exons, the infected cells will likely produce both secreted andmembrane bond forms of IgM (Webb, C. F., et al., J. Immunol.,143:3934-3939 (1989)). Still other methods can be used to detectantibody production. If the-host cell is E. coli, a nitrocelluloseoverlay is possible, and such methods have been frequently used todetect E. coli producing particular proteins (Sambrook, J., et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)). Other methods of detection arepossible and one in particular, which involves the concept of “viralcoating”, is discussed below.

Viral coating can be used as a means of identifying viruses encodingantigen-combining molecules. In this method, a viral vector is used todirect the synthesis of diverse antibody molecules. Upon lytic infectionof host cells, and subsequent cell lysis, the virus becomes “coated”with the antibody product it directs. That is, the antibody moleculebecomes physically linked to the outside of a mature virus particle,which can direct its synthesis. Methods for viral coating are describedbelow. Viruses coated by antibody can be physically selected on thebasis of their affinity to antigen which is attached to a solid support.The number of particles which can be screened using this approach iswell in excess of 10⁹ and it is possible that 10¹¹ different antibodygenes could be screened in this manner. In one embodiment, an affinitymatrix containing antigen used to purify those viruses encoding antibodymolecules with affinity to antigen and which coat the surface of thevirus which encodes those antibodies is used.

One method of viral coating is as follows: A diverse library ofbacteriophage A encoding parts of antibody molecules that are expressedin infected E. coli and which retain the ability to bind antigens iscreated, using known techniques (Orlandi, R., et al., Proc. Natl. Acad.Sci. USA, 86:3833 (1989); Huse, W. D., et al., Science, 246:1275 (1989);Better, M., et al., Science, 240:1041 (1988); Skerra, A. and A.Pluckthon, Science, 240:1038 (1988)). Bacteria infected with phage areembedded in a thin film of semisolid agar. Greater than 10⁷ infectedbacteria may be plated in the presence of an excess of uninfectedbacteria in a volume of 1 ml of agar and spread over a 10 cm² surface.The agar contains monovalent antibody “A” (Parham, P., In Handbook ofExperimental Immunology: Immunochem., Blackwell Scientific Publishers,Cambridge, Mass., pp. 14.1-14.23 (1986)), which can bind the A coatproteins and which has been chemically coupled to monovalent antibody“B”, which can bind an epitope on all viral directed antibody molecules.Monovalent antibodies are used to prevent the crosslinking of viralparticles. Upon lytic burst, progeny phage particles become effectivelycross linked to the antibody molecule they encode. Because lysis occursin semisolid medium, in which diffusion is slow, cross linking between agiven phage and the antibody encoded by another phage is minimized. Anitrocellulose filter (or other protein binding filter) is prepared asan affinity matrix by adsorbing the desired antigen. The filter is thenblocked so that no other proteins bind nonspecifically. The filter isoverlayed upon the agar, and coated phage are allowed to bind to theantigen by way of their adherent antibody molecules. Filters are washedto remove nonspecifically bound phage. Specifically bound phagetherefore represent phage encoding antibodies with the desiredspecificity. These can now be propagated by reinfection of bacteria.

Thus the present invention makes it possible to produce antigen-bindingmolecules which, like antibodies produced by presently-availabletechniques, bind to a selected antigen (i.e., having binding specifity).Antibodies produced as described can be used, for example, to detect andneutralize antigens and deliver molecules to antigenic sites.

EXAMPLE I Amplification of IgM Heavy Chain Variable Region DNA from mRNA

IgM heavy chain variable DNA is amplified from mRNA by the procedurerepresented schematically in FIG. 2. In FIG. 2, Panel A depicts therelevant regions of the poly adenylated mRNA encoding the secreted formof the IgM heavy chain. In Panel A, S denotes the sequences encoding thesignal peptide which causes the nascent peptide to cross the plasmamembrane, a necessary step in the processing and secretion of theantibody. V, D and J derive from separate exons and together comprisethe variable region. C_(H)1, C_(H)2, and C_(H)3 are the three constantdomains of Cμ. “Hinge” encodes the hinge region. C, B and Z areoligonucleotide PCR primers used in the amplification process. The onlyconstraints on Primers B and Z are that they are complementary to themRNA, and occur in the order shown relative to C. Primer C, in additionto being complementary to mRNA, has an extra bit of sequence at its 5′end which allows the cloning of its PCR product. This is describedbelow. Panel B depicts the reverse transcript DNA product of the mRNAprimed by oligonucleotide Z, with the addition of poly-dC by terminaltransferase at the 3′ end of the product. Panel C depicts the annealingof primer A to the reverse transcript DNA represented in Panel B. PrimerA contains the restriction endonuclease site RE1, with additional DNA atits 5′ end. The constraints on the RE1 site are described in Example 2.Panel D depicts the final double stranded DNA PCR product made utilizingprimers A and B. Panel E depicts the PCR product shown in Panel Dannealed to Primer C. Panel F is a blow up of panel E showing thestructure of primer C. Primer C consists of two parts: a 3′ partcomplementary to IgM heavy chain mRNA as shown, and a 5′ part whichcontains restriction site RE2 and spacer. Constraints on RE2 aredescribed in Example 2. Panel G depicts the final double stranded DNAPCR product utilizing Primers A and C and the product of the previousPCR (depicted in Panel D) as template. The S, V, D, J regions are againdepicted.

EXAMPLE 2 Construction of Heavy Chain Framework Vector

A heavy chain framework vector, designated pFHC, is constructed, usingknown techniques (See FIG. 3). It is useful for introducingantibody-encoding DNA into host cells, in which the DNA is expressed,resulting in antibody production. The circular plasmid (above) isdepicted linearized (below) and its relevant components are shown. Theneomycin antibiotic resistance gene (neo^(R)) is useful for selectingtransformed animal cells (Sambrook, J., et al., Molecular Cloning ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)). The bacterial replication origin andampicillin antibiotic resistance genes, useful respectively, forreplication in E. coli and rendering E. coli resistant to ampicillin,can derive from any number of bacterial plasmids, including PBR322(Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).The Cμ enhancer, which derives from the intron between exons J andC_(H)1 of the Cμ gene, derives from any one of the cloned Cμ genes(Kawakami, T., et al., Nucleic Acids Research, 8:3933 (1980); Honjo, T.,Ann. Rev. Immunol., 1:499 (1983)) and increases levels of transcriptionfrom antibody genes. LTR contains the viral promoter from the MoloneyMLV retrovirus DNA (Mulligan, R. C., Experimental Manipulation of GeneExpression, New York Academic Press, p. 155 (1983)). D represents thePCR primer described in the text, depicted in its 5′ to 3′ orientation.The only constraints on D are its orientation, its complementarity topFHC and its order relative to the RE1 and RE2 cloning sites.Preferably, D is within 100 nucleotides of RE1. The cDNA cloning sitecontains restriction endonuclease sites RE1 and RE2, separated by spacerDNA which allows, their efficient cleavage. The constraints on RE1 andRE2 are described below. The Cμ exons, as described in the text andliterature, direct the synthesis of IgM heavy chain. Only part of C_(H)1is present, as described below. C_(H)3 is chosen to contain the Cμsregion which specifies a secreted form of the heavy chain ((Kawakami,T., et al., Nucleic Acids Research, 8:3933 (1980); Honjo, T., Ann. Rev.Immunol., 1:499 (1983)). Finally, pFHC contains poly A addition andtermination sequences which can be derived from the Cμ gene itself(Honjo, T., Ann. Rev. Immunol., 1:499 (1983); Kawakami, T., et al.,Nucleic Acids Research, 8:3933 (1980)). One potential advantage of usingthe entire Cμ gene is that in some host cell systems, a membrane boundand secreted form of IgM may be expressed (Granowicz, E. S., et al., J.Immunol, 125:976 (1980)).

The plasmid can be produced by combining the individual components, ornucleic acid segments, depicted in FIG. 3, using PCR cassett assembly(See below). Because the entire nucleotide sequence of each component isdefined, the entire nucleotide sequence of the plasma is defined.

The constraints on RE1 are simple. It should be the sole cleavage siteon the plasmid for its restriction endonuclease. The choice of RE1 canbe made by computer based sequence analysis (Intelligenetics Suite,Release 5:35, Intelligenetics).

The constraints on RE2 are more complex. First, it must be the solecleavage site on the plasmid for its restriction endonuclease, asdescribed for RE1. Moreover, the RE2 site must be such that when the PCRproduct is inserted, a gene is thereby created which is capable ofdirecting the synthesis of a complete IgM heavy chain. This limits thechoices for RE2, but the choices available can be determined by computerbased sequence analysis. The choices can be determined as follows.First, a list of restriction endonucleases that do not cleave pFHC iscompiled (see Table 1).

TABLE 1 Non-Cutting Enzymes for the Mouse Cμ Gene AatII AhaII AseI AvrIIBgII BspHI BssHII BstBI ClaI DraI EagI EcoRI EcoRV FspI HgaI HincII HpaIKpnI MluI NaeI NarI NdeI NotI NruI PaeR7I PvuI RsrII SacII SaII ScaISfII SnaBI SpeI SphI SspI StuI TthlllI XbaI XhoI

These are called the “rare non-cutters.” Next, the sequence of C_(H)1 isrewritten with “N” at the third position of each codon and entered intothe computer. This is called the “N-doped sequence” (See FIG. 4). Next,the rare non-cutters are surveyed by computer analysis for those whichwill cleave the N-doped sequence. The search program will show apossible restriction endonuclease site, assuming a match between N andthe restriction endonuclease cutting site. For example, with 39 rarenon-cutters, 22 will cleave the N-doped sequence of Cμ C_(H)1, many ofthem several times (see Table 2). In this table, “Def” means a definitecut site, of which there are none, because of the Ns. “Pos” means apossible cleavage site at the indicated nucleotide position if N ischosen appropriately. “Y” indicates any pyrimidine, “R” indicates anypurine and “N” indicates any nucleotide. The nucleotide positions referto coordinates represented in FIG. 4.

TABLE 2 ENZYME RECOGNITION CUT SITE AatII (GAGGTC) Def none Pos 250 309AhaII (GRCGYC) Def none Pos 247 306 AvrII (CCTAGG) Def none Pos 204BspHI (TCATGA) Def None Pos 138 BsshII (GCGCGC) Def none Pos 189 EcoRI(GAATTC) Def none Pos 195 334 EcoRV (GATATC) Def none Pos 214 HgaI(GACGCNNNNN) Def none (NNNNNNNNNNGCGTC) Pos 284 HincII (GTYRAC) Def nonePos 183 220 HpaI (GTTAAC) Def none Pos 220 KpnI (GGTACC) Def none Pos408 NruI (TCGCGA) Def none Pos 174 193 303 PaeR7 (CTCGAG) Def none Pos190 339 PvuI (CGATCG) Def none Pos 178 ScaI (AGTACT) Def none Pos 209266 284 SpeI (ACTAGT) Def none Pos 131 167 359 SphI (GCATGC) Def nonePos 338 SspI (AATATT) Def none Pos 371 StuI (AGGCCT) Def none Pos 149TthlllI (GACNNNGTC) Def none Pos 212 XbaI (TCTAGA) Def none Pos 338 XhoI(CTCGAG) Def none Pos 190 339

Most of these cleavage sites (about 60%) are compatible with the aminoacids specified by C_(H)1. Therefore, it is possible to mutate C_(H)1 tocreate a unique site for such an enzyme without altering the amino acidsequence incoded by C_(H)1. One sequence which illustrates this is shownbelow:

1) . . . ala   met   gly   cys   leu   ala   arg   asp . . . 2) . . .GCC   ATG   GGC   TGC   CTA   GCC   CGG   GAC . . . 3) . . .GCC   ATG   GGC   TGC   CTA   GCG   CGC   GAC . . .                                    ---------                                      BssHII

Line 1 represents part of the actual amino acid sequence specified bythe mouse Cμ C_(H)1 gene region, and line 2 is the actual nucleotidesequence. By changing the sequence to the indicated nucleotidesunderlined on line 3, a cleavage site for the rare non-cutter BssHII iscreated. The new sequence (containing the BssHII site) GCG CGC stillencodes the identical amino acid sequence. Therefore, the sequence ofthe primer C is chosen to be the complement of line 3, and RE2 is theBssHII site. Such a primer will function in the PCR and vectorconstruction as desired. Other examples are possible, and the sameprocess can be used in designing vectors and primers for cloning lightchain variable regions.

The choice for primer C puts a constraint on pFHC. In the example shown,the C_(H)1 region contained on pFHC must begin at its 5′ end with themutant sequence GCG CGC Such mutant fragments can be readily made by theprocess of PCR cassette assembly described below.

The process of PCR cassette assembly is a method of constructing plasmidmolecules (in this case the plasmid pFHC) from fragments of DNA of knownnucleotide sequence. One first compiles a list of restrictionendonucleases that do not cleave any of the fragments. Each fragment isthen individually PCR amplified using synthesized oligonucleotideprimers complementary to the terminal sequences of the fragment. Theseprimers are synthesized to contain on their 5′ ends restrictionendonuclease cleavage sites from the compiled list. Thus, each PCRproduct can be so designed that each fragment can be assembled one byone into a larger plasmid structure by cleavage and ligation andtransformation into E. coli. Using this method, it is also possible tomake minor modifications to modify the terminal sequence of the fragmentbeing amplified. This is done by altering the PCR primer slightly sothat a mismatch occurs. In this way it is possible to amplify the Cμgene starting precisely from the desired point in C_(H)1 (as determinedby oligo C above) and creating the RE2 endonuclease cleavage site.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

6 1 313 DNA Artificial Sequence Synthesized Nucleotide Sequence 1 agtcag tcc ttc cca aat gtc ttc ccc ctc gtc tcc tgc gag agc ccc 48 Ser GlnSer Phe Pro Asn Val Phe Pro Leu Val Ser Cys Glu Ser Pro 1 5 10 15 ctgtct gat aag aat ctg gtg gcc atg ggc tgc cta gcc cgg gac ttc 96 Leu SerAsp Lys Asn Leu Val Ala Met Gly Cys Leu Ala Arg Asp Phe 20 25 30 ctg cccagc acc att tcc ttc acc tgg aac tac cag aac aac act gaa 144 Leu Pro SerThr Ile Ser Phe Thr Trp Asn Tyr Gln Asn Asn Thr Glu 35 40 45 gtc atc cagggt atc aga acc ttc cca aca ctg agg aca ggg ggc aag 192 Val Ile Gln GlyIle Arg Thr Phe Pro Thr Leu Arg Thr Gly Gly Lys 50 55 60 tac cta gcc acctcg cag gtg ttg ctg tct ccc aag agc atc ctt gaa 240 Tyr Leu Ala Thr SerGln Val Leu Leu Ser Pro Lys Ser Ile Leu Glu 65 70 75 80 ggt tca gat gaatac ctg gta tgc aaa atc cac tac gga ggc aaa aac 288 Gly Ser Asp Glu TyrLeu Val Cys Lys Ile His Tyr Gly Gly Lys Asn 85 90 95 aga gat ctg cat gtgccc att cca g 313 Arg Asp Leu His Val Pro Ile Pro 100 2 104 PRTArtificial Sequence Synthesized Peptide 2 Ser Gln Ser Phe Pro Asn ValPhe Pro Leu Val Ser Cys Glu Ser Pro 1 5 10 15 Leu Ser Asp Lys Asn LeuVal Ala Met Gly Cys Leu Ala Arg Asp Phe 20 25 30 Leu Pro Ser Thr Ile SerPhe Thr Trp Asn Tyr Gln Asn Asn Thr Glu 35 40 45 Val Ile Gln Gly Ile ArgThr Phe Pro Thr Leu Arg Thr Gly Gly Lys 50 55 60 Tyr Leu Ala Thr Ser GlnVal Leu Leu Ser Pro Lys Ser Ile Leu Glu 65 70 75 80 Gly Ser Asp Glu TyrLeu Val Cys Lys Ile His Tyr Gly Gly Lys Asn 85 90 95 Arg Asp Leu His ValPro Ile Pro 100 3 313 DNA Artificial Sequence Synthesized NucleotideSequence 3 agncantcnt tnccnaangt nttnccnctn gtntcntgng anagnccnctntcnganaan 60 aanctngtng cnatngcntg nctngcncgn ganttnctnc cnagnacnatntcnttnacn 120 tgnaantanc anaanaanac ngangtnatn canggnatna gnacnttnccnacnctnagn 180 acnggnggna antanctngc nacntcncan gtnttnctnt cnccnaanagnatnctngan 240 ggntcngang antanctngt ntgnaanatn cantanggng gnaanaanagnganctncan 300 gtnccnatnc cng 313 4 8 PRT Artificial SequenceSynthesized Nucleotide Sequence 4 Ala Met Gly Cys Leu Ala Arg Asp 1 5 524 DNA Artificial Sequence Synthesized Nucleotide Sequence 5 gccatgggctgcctagcccg ggac 24 6 24 DNA Artificial Sequence Synthesized NucleotideSequence 6 gccatgggct gcctagcgcg cgac 24

What is claimed is:
 1. A diverse library of DNAs encoding families ofantigen-combining proteins produced by the method of: (a) obtaining DNAcontaining genes encoding antigen-combining proteins; (b) combining theDNA containing genes encoding antigen-combining proteins with sequencespecific primers which are oligonucleotides homologous to conservedregions of the genes; (c) performing sequence specific geneamplification that results in amplified genes; and (d) introducing theamplified genes into a framework antibody vector.
 2. Host cellscomprising DNA selected from the library of DNAs of claim
 1. 3. The hostcells of claim 2 wherein the host cells are prokaryotic.
 4. The hostcells of claim 2 wherein the host cells are eukaryotic.
 5. The hostcells of claim 4 wherein the host cells are immortalized culturedmammalian cells.
 6. The host cells of claim 5 wherein the immortalizedcultured mammalian cells are myelomas or plasmacytomas.
 7. The hostcells of claim 2 wherein said DNA is introduced into host cells by amethod selected from the group consisting of: electroporation, calciumphosphate coprecipitation, protoplast fusion, viral infection, and cellfusion.
 8. The host cells of claim 2 wherein the library of DNAsencoding families of antigen-combining proteins is contained in anexpression vector.
 9. The host cells of claim 2 wherein the DNAsencoding families of antigen-combining proteins encode antigen-combiningproteins selected from the group consisting of: immunoglobulin heavychain variable regions and immunoglobulin light chain variable regions.10. The host cells of claim 9 wherein DNAs encoding immunoglobulin heavychain variable regions are introduced simultaneously with orsequentially to DNAs encoding immunoglobulin light chain variableregions.
 11. The host cells of claim 2 wherein the host cells produceantigen-combining molecules of selected specificity.
 12. A diverselibrary of DNAs encoding families of antibodies produced by a methodcomprising: (a) obtaining DNA comprising genes encoding antibodies; (b)combining said DNA with sequence specific primers which areoligonucleotides homologous to conserved regions of the genes; (c)performing sequence specific gene amplification that results inamplified genes; and (d) introducing the amplified genes into aframework antibody vector.
 13. The library of claim 12 wherein said DNAcomprises a vector.
 14. The library of claim 13, wherein said vector isan expression vector.
 15. The library of claim 12 wherein said DNA isobtained from antibody-producing cells.
 16. The library of claim 12wherein said DNA is obtained from cells selected from the groupconsisting of: spleen cells; peripheral blood cells; lymph nodes;inflammatory tissue cells; bone marrow cells; B cells; T cells; humancells; and non-human cells.
 17. The library of claim 12 wherein thesequence specific gene amplification comprises the polymerase chainreaction.
 18. The library of claim 12 wherein the sequence specific geneamplification comprises rolling circle amplification.